Successful treatment of hepatic veno-occlusive disease after myeloablative allogeneic hematopoietic stem cell transplantation by early administration of a short course of methylprednisolone.

Hepatic veno-occlusive disease (VOD) is one of the most common and important regimen-related toxicities observed after hematopoietic stem cell transplantation (HSCT). There are no universally accepted preventative or therapeutic approaches for VOD. We prospectively evaluated the safety and efficacy of a short course of methylprednisolone (MP) in 48 patients undergoing allogeneic HSCT who were diagnosed with hepatic VOD. MP was administered at a dose of 0.5 mg/kg i.v. every 12 h for a total of 14 doses, and then discontinued without taper. Thirty (63%) patients responded with a reduction in total serum bilirubin of 50% or more after 10 days of treatment. In univariate analysis, non-responders had a higher total bilirubin at the start of MP therapy, more weight gain, evidence of fungal infection and platelet refractoriness. High SGPT and early engraftment were significant factors among responders. Twenty-five of the 30 responders survived up to day +100, whereas all but three non-responders died within 100 days post-HSCT, for a probability of survival of 58% among responders and 10% for non-responders. Prospective comparative studies are needed to confirm the observed encouraging outcome of MP therapy for VOD.

Immunodominant minor histocompatibility antigens expressed by mouse leukemic cells can serve as effective targets for T cell immunotherapy.

Numerous minor histocompatibility antigens (MiHAs) show tissue-specific expression and can induce vigorous T cell responses. They therefore represent attractive targets for leukemia immunotherapy mediated by adoptive transfer of T cells. The main objective of this work was to determine whether MiHAs expressed by normal hematopoietic cells were present on leukemic cells and whether they could trigger lysis by cytotoxic T lymphocytes (CTLs). CTL assays showed that mouse leukemic cells of both lymphoid and myeloid lineages were sensitive to CTLs targeted toward some but not all MiHAs. In four out of four strain combinations in which we primed CTLs against immunodominant MiHAs, effectors killed leukemic blasts, whereas no cytotoxicity was observed when CTLs were targeted toward four immunorecessive MiHAs. Testing of HPLC fractions obtained from normal and leukemic cells provided molecular evidence that leukemic blasts expressed only some of the MiHAs found on normal mouse hematopoietic cells. Decreased density of H-2 class I molecules at the surface of leukemic cells suggests that down-regulation of genes encoding either class I molecules or proteins involved in antigen processing played a role in the aberrant expression of MiHAs. In vivo resistance to the leukemic cells by various strains of mice correlated with in vitro CTL activity. These results show that leukemic cells express only some (immunodominant) MiHAs and suggest that this subset of MiHAs represent prime targets for adoptive immunotherapy.

Distinct patterns of minimal residual disease associated with graft-versus-host disease after allogeneic bone marrow transplantation for chronic myelogenous leukemia.

PURPOSE: Allogeneic bone marrow transplantation (BMT) has been shown to provide effective therapy for chronic myelogenous leukemia (CML), but previous reports have also demonstrated the persistence of bcr-abl-positive cells for months to years after BMT in the majority of patients. To evaluate the biologic significance of persistent bcr-abl-positive cells, we examined the relationship between clinical parameters known to affect the risk of relapse and the ability to detect bcr-abl-positive cells post-BMT. PATIENTS AND METHODS: We analyzed 480 samples from 92 patients at two transplant centers for the presence of bcr-abl-positive cells by polymerase chain reaction (PCR). Two different BMT preparative regimens and protocols for prevention of graft-versus-host disease (GVHD) were used. One center used cyclophosphamide plus total-body irradiation (CY/TBI) and T-cell-depleted marrow; the second center used busulfan plus cyclophosphamide (Bu/CY) and untreated marrow with cyclosporine and methotrexate (Csp/MTX) as GVHD prophylaxis. RESULTS: We first determined the percent of patients at each center with > or = one PCR-positive (PCR+) result at defined intervals post-BMT. Between 0 and 6 months post-BMT, the majority of patients (80% to 83%) in both populations had PCR-detectable bcr-abl-positive cells. Between 6 and 24 months post-BMT, 80% to 88% of patients who received T-cell-depleted marrow remained PCR+, as compared with 26% to 30% of patients who received unmodified marrow. After 24 months post-BMT, the percentage of PCR+ patients was not significantly different in the two populations. This pattern of detection of bcr-abl-positive cells post-BMT followed the development of chronic GVHD in patients who received unmodified marrow. All patients were also divided into three groups based on post-BMT PCR results as follows: (1) persistent PCR+ (n = 29), (2) intermittent PCR-negative ([PCR-] n = 40), and (3) persistent PCR- (n = 23). These three groups were found to have a low, intermediate, and high probability of maintaining remission and disease-free survival, respectively (P = .0001). Intermittent or persistent PCR- results, which reflect levels of minimal residual disease < or = the limit of detection by PCR, were clearly associated with both acute (P = .004) and chronic (P = .000005) GVHD. Nevertheless, 44% of patients without GVHD also had intermittent or persistent PCR- assays. CONCLUSION: The persistence of PCR-detectable bcr-abl-positive cells early post-BMT in more than 80% of patients suggests that neither BMT preparative regimen effectively eradicates CML cells in most patients. Subsequently, acute and/or chronic GVHD are associated with a decreased ability to detect residual bcr-abl-positive cells, which suggests that immunologic mechanisms mediated by donor cells are important for inducing long-term remissions after BMT. The demonstration that 44% of patients without GVHD had either low or undetectable levels of residual leukemia suggests the presence of mechanisms capable of suppression or eradication of CML independent of GVHD.

Thymic and extrathymic differentiation and expansion of T lymphocytes following bone marrow transplantation in irradiated recipients.

Thymic function is severely impaired in most marrow transplant recipients. To evaluate the impact of thymic hypoplasia on T cell reconstitution following marrow transplantation, we compared the phenotype and function of T lymphocytes in thymectomized recipients with those of euthymic hosts. Irradiated C57BL/6 mice (Thy1.2+, Ly5.1+) received 10(7) T cell-depleted B6.Ly5.2 bone marrow cells (Thy1.2+, Ly5.2+), with or without 3 x 10(5) B6.PL lymph node cells (Thy1.1+, Ly5.1+) as a source of T lymphocytes. Multiparameter flow cytometry analysis showed that in euthymic mice (group 1), T cell reconstitution was carried out by donor hematopoietic stem cells that differentiated in the host's thymus, whereas the production of chimeric T cells in athymic recipients depended on the presence or absence of T cells in the graft. When T lymphocytes were present in the graft (group 2), their progeny constituted the vast majority of splenic T cells on day 100 posttransplant. When the graft did not contain T lymphocytes (group 3), T cell reconstitution resulted from extrathymic maturation of donor hematopoietic progenitors; T cells differentiating along this pathway expressed lower levels of T cell receptor and a large proportion of the CD8+ subset expressed CD8alpha alpha homodimers. The T cell receptor Vbeta profile of all chimeras was similar to that of normal C57BL/6 mice. Compared with T cells found in euthymic recipients, those in mice from groups 2 and 3 were less abundant (particularly with respect to the CD4+ subset), displayed the CD44/CD45 phenotype of activated memory cells, and expressed high levels of IL-2 receptor beta chain. These results show that both the presence or absence of the thymus and the composition of the grafted inoculum determine the source and extent of posttransplant T cell reconstitution. Because they determine the nature of the differentiation pathway taken during T cell development in the host, these two factors can exert a critical influence on the appearance of graft vs. host disease and the level of host immunocompetence.

Quantitative assessment of hematopoietic chimerism after allogeneic bone marrow transplantation has predictive value for the occurrence of irreversible graft failure and graft-vs.-host disease.

Primary graft failure, secondary to either host-vs.-graft reaction or delayed engraftment, and graft-vs.-host disease (GVHD) are among the most difficult clinical problems to manage in the field of allogeneic bone marrow transplantation (BMT). Early diagnosis of both conditions would greatly improve their outcome. Using fluorescence in situ hybridization (FISH) with an X- and Y-probe mixture, we sequentially monitored chimerism of neutrophils and lymphoid cells from day 1 to 100 in 28 consecutive recipients of sex-mismatched unmanipulated bone marrow grafts. The objective was to quantitatively assess the evolution of chimerism during this crucial time interval and to determine whether chimerism patterns would be predictive of engraftment and GVHD. In recipients with primary graft failure (n=7), the presence of donor-type neutrophils and NK cells as well as the predominance of donor-type T cells distinguished patients who responded to G-CSF (n=5) from nonresponders (n=2). Furthermore, the clearance of host CD3+CD56- cells during days 5-10 posttransplantation was significantly hastened in patients who subsequently developed acute (delta=80%) or chronic (delta=81%) GVHD compared with patients without GVHD (delta=17%). Thus, our data suggest that molecular monitoring of the fate of host/donor hematopoietic cells in the early posttransplantation period could be useful in differentiating patients with delayed engraftment from those with irreversible rejection and in predicting the occurrence of GVHD as soon as day 10. This investigational approach may provide an appropriate basis on which to select adequate treatment for primary graft failure and high-risk candidates that could benefit from novel preemptive therapies for GVHD.

Interstitial deletion of the long arm of chromosome 5 (5q-) in leukemia and other hematological disorders: clinical and biological relevance of variable breakpoint patterns.

From January 1983 to January 1985, we have found a deletion of the long arm of chromosome 5 (5q-) in the bone marrow cells of 8 patients; they represent consecutive patients referred to our institution for investigation and treatment of the following hematological disorders in whom the 5q- abnormality was found: acute nonlymphocytic leukemia (ANLL) (3), refractory anemia with excess blasts (RAEB) (2), preleukemia (PL) (1), sideroblastic anemia (SA) (1), refractory anemia (RA) (1). The deletion proved to be interstitial in all patients, with 3 different breakpoint patterns emerging: q13q33, q11.2q21, q11.2q33. Proximal breakpoint q11.2 was found only in patients with an initial diagnosis of ANLL. The common region deleted in all patients was comprised between bands q13q21. The clinical relevance and biological meaning of the heterogeneity of proximal and distal breakpoints found in this study are discussed.

Immunobiology of allogeneic peripheral blood mononuclear cells mobilized with granulocyte-colony stimulating factor.

The use of mobilized peripheral blood (PB) stem cells for autologous transplantation initially generated much enthusiasm because of enhanced engraftment in comparison to marrow stem cells and avoidance of general anesthesia for the donor. Its application to the allogeneic setting seemed inevitable. For obvious ethical reasons, allogeneic donors are mobilized with cytokines only, mainly granulocyte colony-stimulating factor (G-CSF). Results from preliminary studies suggest that in comparison to standard bone marrow transplants, outcomes such as engraftment, host-versus-graft reaction, graft-versus-host disease, graft-versus-leukemia and immunological reconstitution may be different. Surprisingly, G-CSF, previously recognized as a late acting lineage-specific factor for neutrophil production, not only disrupts homeostasis between stem cells and their microenvironment, but also induces significant quantitative and qualitative changes in the accessory cell compartment, affecting lymphocytes, monocytes, natural killer, dendritic, and stromal cells. Furthermore, mobilization of huge numbers of non-professional antigen presenting cells (CD34+ stem cells) amplifies the tolerizing potential of PB stem cell grafts. Thus, G-CSF mobilization provides PB transplants with different immunobiologic properties in comparison to standard bone marrow grafts. Whether these immunobiologic differences will lead to better transplant outcomes remains to be shown through much awaited results of large randomized clinical trials.

The mechanism of graft-host-tolerance in murine radiation chimeras transplanted across minor histocompatibility barriers.

A better understanding of the mechanism(s) involved in graft-host-tolerance following allogeneic bone marrow transplantation is needed to develop new strategies to prevent graft-versus-host disease (GVHD). Based on previous studies, mainly in MHC-mismatched donor-recipient pairs, three hypotheses have been proposed: clonal deletion, active suppression and lack of adequate antigen-presenting cells. Our goal was to identify the mechanism(s) by which tolerance is achieved and maintained in radiation chimeras transplanted across minor histocompatibility barriers. Healthy (B6----LP) chimeras were obtained following injection of 10(7) C57BL/6 marrow cells to irradiated (9.5 Gy) LP hosts and used experimentally 100 days after chimerization. The tolerance state of (B6----LP) chimeras could not be abrogated after i.v. transfer of 5 x 10(7) donor-type spleen cells alone or with repeated i.p. injection of host-type antigen-presenting cells. No GVHD was observed when 10(7) marrow cells plus 5 x 10(7) spleen cells from (B6----LP) chimeras were injected to irradiated LP recipients. Chimera spleen cells suppressed GVHD when adoptively transferred to LP recipients of a C57BL/6 graft. These results suggest that in this model the presence of suppressor cells is both necessary and sufficient to maintain graft-host-tolerance.

Studies of immunologic tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation in mice.

We showed previously that transplantation of 10(7) unmanipulated C57BL/6 marrow cells to irradiated LP mice yields healthy (B6-LP) chimeras showing no signs of rejection or graft-versus-host disease (GVHD). The aim of this work was to gain more insight into the mechanism(s) responsible for tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation (BMT). (B6-LP) chimeras showed very good immune reconstitution when studied in vitro for proliferative response to mitogens and alloantigens and generation of T cell cytotoxic activity. In co-culture experiments their spleen cells showed no natural suppressor activity. When used as cell donors, their capacity to initiate GVHD in four strains of mice presenting H-2 differences was normal when compared to C57BL/6 donors. However, they provoked no GVHD in the three strains of H-2 compatible mice studied. Re-irradiated (B6-LP) chimeras rapidly died of GVHD following injection of C57BL/6 marrow + spleen cells. (B6-LP.R111) chimera cells appeared tolerant to LP minor antigens presented in the context of H-2r or H-2b. No anamnestic anti-idiotypic suppressor response was noted when stable (B6-LP) chimeras were stimulated with naive C57BL/6 cells. These findings suggest that in BMT chimeras transplanted across minor histocompatibility barriers: (1) both host and donor-derived antigen-presenting cells can present host antigens to donor T cells whose numbers in the marrow inoculum will determine if GVHD or tolerance will ensue, (2) GVHD can be triggered by only a limited number of 'dominant' minor antigens, and (3) we found no evidence for the presence of natural suppressors, veto cells or anti-idiotypic suppressor T cells.

Phenotypic and functional characterization of peripheral blood and bone marrow natural killer cells prior to autologous transplantation.

NK cells can exert potent anti-leukemia activity after either autologous or allogeneic BMT. However, in autologous blood or marrow transplant patients, NK cell number and/or function could be reduced, and also may vary according to the sampling site. In order to evaluate the hypothesis that blood or marrow grafts from autologous transplant patients exhibit impaired NK cell activity that could contribute to disease recurrence, we evaluated the immunologic characteristics of NK cells in the bone marrow (BM) and peripheral blood (PB) from 27 patients undergoing autologous BMT, and also from 20 normal donors. We measured baseline and interleukin-2 (IL-2)-activated NK cell cytotoxicity, as well as expression of IL-2 receptors (IL-2R) (alpha-chain (p55) and beta-chain (p75)), and adhesion molecules. The cytotoxic activity of PB NK cells was significantly lower in autologous transplant patients than in normal donors (P < 0.0005) and this difference was not mitigated following IL-2 activation. In contrast, BM from autologous patients showed normal NK cell cytotoxicity, but contained higher numbers of NK cells (P < 0.025), with more intense CD56 expression (P < 0.05). Expression of p75 was lower on BM than on PB NK cells in both patients and normal donors. In addition, induction of p55 by IL-2 was abrogated in autologous PB NK cells. Therefore, depending on the site of harvest and the nature of donor cells (pre-BMT vs normal), our results show significant differences in NK cell number, function, and IL-2 receptor expression. This may affect relapse rates following autologous transplants performed with either PB or BM grafts.

Sequential analysis of early hematopoietic reconstitution following allogeneic bone marrow transplantation with fluorescence in situ hybridization (FISH).

Using in situ hybridization with an X and Y chromosome probe mixture, we have sequentially studied peripheral blood samples from 10 patients (four males/six females) in an HLA-matched allogeneic setting in order to monitor the kinetics of early hematopoietic reconstitution. Interphase cells from smears consisting of purified granulocytic and lymphocytic populations respectively were studied in three patients at 24, 48, 72 and 96 h post-transplant. This period was arbitrarily defined as the immediate post-transplant period. These three patients plus seven others were studied sequentially at days 5, 10, 15, 20, 25 and 50 post-transplant, defined as the intermediate post-transplant period. The X and Y probes were indirectly labelled with rhodamine and fluoresceine isothiocyanate, respectively. Donor neutrophils were detected as early as 24 h post marrow infusion followed by a significant expansion at 48 h. At 96 h post-transplant, the median percentage of donor neutrophils was > 90%. In the immediate post-transplant period, most of the lymphocytes were of recipient origin. However, we have documented a significant expansion in donor lymphocytes, starting at day 5 post-transplant in most patients. Almost complete chimerism for the myeloid and lymphoid lineages was established at days 10 and 25 post-transplant, respectively. All patients engrafted normally according to standard clinical criteria. Follow-up data for those surviving > or = 100 days (eight patients), showed persistence of this pattern of hematopoietic reconstitution in all but one patient. Molecular monitoring of early engraftment has enabled us to unravel a distinct biphasic pattern of myeloid and lymphoid engraftment.

Mycobacterium tuberculosis infection in allogeneic bone marrow transplantation patients.

Bone marrow transplant (BMT) recipients are prone to bacterial, viral and fungal infections. Mycobacterium tuberculosis infection can occur in these patients, but the incidence is lower than that of other infections. This report describes four patients with Mycobacterium tuberculosis infection identified from 641 adult patients who received a BMT over a 12-year period (prevalence 0.6%). The pre-transplant diagnosis was AML in two patients and CML in the other two. Pre-transplant conditioning consisted of BU/CY in three patients and CY/TBI in one. Graft-versus-host disease (GVHD) prophylaxis was MTX/CsA in three patients and T cell depletion of the graft in one patient. Sites of infection were lung (two), spine (one) and central nervous system (one). Onset of infection ranged from 120 days to 20 months post BMT. Two patients had co-existing CMV infection. One patient had graft failure. The two patients who received anti-tuberculous (TB) therapy recovered from the infection. Although the incidence of tuberculosis in BMT patients is not as high as in patients with solid organ transplants, late diagnosis due to the slow growth of the bacterium can lead to delay in instituting anti-TB therapy. A high index of suspicion should be maintained, particularly in endemic areas.

Hepatitis B virus reactivation with clinical flare in allogeneic stem cell transplants with chronic graft-versus-host disease.

We conducted a retrospective study with the aim of identifying risk factors and clinical characteristics associated with HBV reactivation and clinical flare after allogeneic stem cell transplantation (aSCT). We reviewed the King Faisal Specialist Hospital and Research Center International Bone Marrow Transplant Registry database from January 1998 to June 2000. Complete serological screening for HBV was available in 128 of 131 patients transplanted during that period. Fifty-four (42%) had evidence of prior infection and recovery from HBV before transplant (hepatitis B core antibody positive, B surface antigen negative). Forty-two were evaluable for HBV reactivation and clinical flare. Six (14%) reactivated with clinical flare as documented by seroconversion and/or positive HBV DNA in the serum with biochemical hepatitis at 5.5, 18, 18, 19, 21 and 23 months post-transplant. Five of fifteen patients with chronic graft-versus-host disease (cGVHD) reactivated with clinical flare in contrast to 1/27 without cGVHD (RR: 9.0, 95% CI: 1.2-70.1 P < 0.02). HBV reactivation with clinical flare occurred during immunosuppressive therapy tapering or withdrawal in all patients. In conclusion, hepatitis B core antibody positive allogeneic stem cell recipients with cGVHD are at significant risk of HBV reactivation with clinical flare.

Extramedullary myeloid cell tumours localised to the mediastinum: a rare clinicopathological entity with unique karyotypic features.

Extramedullary myeloid cell tumour (EMMT) localised to the mediastinum is a rare manifestation of acute myeloid leukaemia, forming less than 4% of all cases of EMMT. In contrast to other types of EMMT, cytogenetic characteristics of this rare entity are relatively unknown. This report describes a patient with EMMT who had evidence of superior vena cava syndrome and normal peripheral blood counts at diagnosis. The results from an initial biopsy specimen were consistent with a diagnosis of mediastinal large B cell lymphoma. A diagnosis of acute myeloid leukaemia was made three months after initial diagnosis by bone marrow examination. Review of the initial biopsy specimen showed strong positivity for myeloperoxidase, revealing that the patient had been initially misdiagnosed as having large B cell lymphoma. Cytogenetic studies revealed a near triploid and near tetraploid karyotype with structural abnormalities in 12 and three metaphases, respectively. Review of the literature showed that a near tetraploid or triploid karyotype is found in most of the reported cases of mediastinal EMMT. Thus, the presence of a near triploid/tetraploid karyotype and mediastinal EMMT may represent a specific subset of EMMT. The biological relevance of this observation is discussed.

Vibrissa-related behavior in mice: transient effect of ablation of the barrel cortex.

Knowing that the mystacial vibrissae are an important part of the tactile sensory apparatus of rodents, we investigated the role of the barrel cortex - the endstation of the pathway between whiskerpad and cerebral cortex - in mouse behavior. We tested 15 female adult mice 2 and 10 weeks after both unilateral ablation of the barrel cortex and removal of the vibrissae on the same side in order to assess acute as well as transient effects of the cortical lesion. Two kinds of behavioral tests were performed on animals permanently provided with opaque lenses: one involved a passive stimulation of the vibrissae; the other was the 'gap-crossing' test which required the animal's active use of the vibrissae. Lesioned subjects did not show a deficit during passive stimulation of the vibrissae. On the contrary, there was a deficit during the gap-crossing test 2 weeks after the ablation of the barrel cortex. The deficit partly disappeared when the subjects were tested 10 weeks later. The results show that in mice, the barrel cortex is involved in the performance of complex behavioral tasks. The recovery of function could be due to changes in strategies to solve the gap-crossing test and/or to physical changes in neuronal circuitry. In either case, the results are relevant for the interpretation of cortical transplantation models using the whisker-to-barrel pathway.

14q+ abnormality with probable t(8;14)(q24;q32) in a young Haitian immigrant with acquired immunodeficiency syndrome and concomitant Burkitt's-like lymphoma.

Cytogenetic analyses were carried out in a young Haitian immigrant with acquired immunodeficiency syndrome and concomitant Burkitt's-like lymphoma associated with massive bone marrow infiltration. A characteristic 14q+ abnormality was found in all bone marrow cells examined. Although chromosome abnormality involving band 8q24 was not evident in all the cells examined, some karyotypes show that the typical t(8;14)(q24;q32) is most probably present. No other complex rearrangements could be identified. This is the first report of concomitant acquired immunodeficiency syndrome and Burkitt's-like lymphoma in the Haitian community. Our cytogenetic findings provide further evidence for the role of specific chromosomal rearrangements in Burkitt's-like lymphoma oncogenesis in the setting of acquired immunodeficiency syndrome.

Two different schedules for integrating filgrastim as adjuvant therapy in the treatment of patients with advanced stage Hodgkin's lymphoma receiving MOPP/ABV hybrid chemotherapy.

PURPOSE: Management of advanced-stage Hodgkin's disease with a MOPP/ABV hybrid regimen (mechlorethamine, vincristine, procarbazine, prednisone, Adriamycin, bleomycin and vinblastine) has yielded a high complete response rate (75-85%). However, myelosuppression can limit delivery of treatment. Filgrastim has been shown to reduce chemotherapy-related neutropenia and allow for on-time administration of planned doses of chemotherapeutic agents. The objective of this study was to find the best way to integrate filgrastim with the MOPP/ABV hybrid regimen. METHODS: Enrolled in this study were 24 patients (aged 18-52 years) with newly diagnosed, histologically documented Hodgkin's disease. In schedule I, patients received filgrastim (5 microg/kg s.c. daily) beginning on day 9, 24 h after administration of ABV. In schedule II, patients received filgrastim concomitantly with procarbazine on days 2-7 (starting 24 h after day-1 MOPP administration and stopping 24 h before ABV administration) as well as after ABV beginning on day 9. Filgrastim after ABV administration was administered until two consecutive ANC readings of 10 x 10(9)/l were achieved. RESULTS: All patients were able to complete all six cycles of therapy. There was a trend to fewer dose reductions in schedule II (0.76%) as compared to schedule I (4.2%) with a P-value of 0.077 (chi-squared test). Specifically, 11.6% of MOPP courses and 5.5% of ABV courses were dose-reduced in schedule I versus 1.7% and 1.4%, respectively, in schedule II. CONCLUSION: In conclusion, filgrastim was effective in supporting the delivery of the MOPP/ABV chemotherapy. Concomitant administration of filgrastim with procarbazine (days 2-7) appears to be safe and allows the maximum dose intensity of this therapy.

A phase I trial of standard and cyclophosphamide dose-escalated CHOP with granulocyte colony stimulating factor in elderly patients with non-Hodgkin's lymphoma.

The purpose of this study was to assess the safety and feasibility of using standard and escalated doses of cyclophosphamide with doxorubicin, vincristine and prednisone (CHOP) plus granulocyte colony stimulating factor (G-CSF) to treat elderly patients who have advanced stage intermediate grade lymphoma. Consenting patients age > or = 65 years who had an acceptable performance status and adequate cardiac, renal and liver function were eligible for this Phase I study. G-CSF, 5 ug per kg, was given daily with each cycle from day 2 until neutrophil recovery of > or = 10 x 10(9)/L. Ten patients received standard CHOP; sequential cohorts of 5 patients were then to be given CHOP with cyclophosphamide doses of 900, 1050, 1200, and 1350 mg/m2. If 2 patients had dose limiting toxicity, cohorts were expanded to 10 patients; if 3 patients within a cohort had dose limiting toxicity, the previous dose level was considered the maximum tolerated dose of cyclophosphamide. Secondary outcomes were average relative received dose intensity, response, progression-free and overall survival, toxicity, hospitalizations and transfusions. Eight patients (80%) completed 6 cycles of standard CHOP plus G-CSF. Therapy was stopped prematurely in 2 patients due to pneumonia (1) and disease progression (1). Six of 11 patients (55%) given CHOP with cyclophosphamide 900 mg/m2 (CHOP-900) completed treatment. Therapy was stopped in 5 patients due to a toxic death from infection (1), cumulative fatigue (3), and pneumonitis (1). Further dose escalations were not attempted due to the inability to complete 6 treatment cycles in 45% of CHOP-900 cases. The received dose intensities of cyclophosphamide relative to standard CHOP measured over the actual time on therapy were 96% with standard CHOP and 115% with CHOP-900. At 3 years, progression free survival is 40% with standard CHOP and 82% with CHOP-900; overall survivals are 40% and 91% respectively. Neutropenia of < 1.0 x 10(9)/L occurred in 47% of treatment cycles with standard CHOP and in 77% with CHOP-900. In both groups, the mean duration of neutropenia was < 2 days. From these studies we conclude that, standard CHOP with G-CSF can be safely given to elderly patients. Escalating the dose of cyclophos

Monitoring of donor/recipient T-cell engraftment kinetics in myeloablative allogeneic stem cell transplantation using short tandem repeat amplification from cell lysates.

Molecular monitoring of donor/recipient T-cell kinetics early post-transplant can provide clues to the immunological events that govern host-versus-graft reaction (HVGR) and graft versus-host-disease (GVHD). We have previously used fluorescence in situ hybridization (FISH) with X and Y probes to monitor recipient T (R-T) cell clearance early after myeloablative allogeneic stem cell transplantation (ASCT). We demonstrated that impaired clearance of residual host-T-cells in the early days post-transplant was associated with graft rejection, while enhanced clearance could be an indicator of increased donor anti-host alloreactivity and predictive of acute GVHD. Although FISH is the most accurate quantitative molecular tool for the determination of the exact donor/recipient-T-cell numbers at any time points post-transplant, it has the disadvantage of being limited to sex mismatched donor/recipient pairs. Our goal was to develop a molecular approach that, irrespective of gender, would be comparable to FISH in accurately determining host residual T-cell clearance after myeloablative conditioning for ASCT. We have genotyped DNA from cell lysates using polymerase chain reaction (PCR) amplification of short tandem repeats (STR) with fluorescently labeled oligonucleotide primers, and used the Genescan 672 software for accurate quantitative analysis of the amplified alleles. Here, we show that this approach allowed us to achieve in T-cells accurate quantitative analyses of amplified donor/recipient alleles in sex matched patients on days +5, +8 and +12 post-transplant, despite severe leukopenia.

Audience effects on alarm calling in chickens (Gallus gallus).

The male domestic chicken (Gallus gallus) has been found to modulate the production of vocal signals in response to the presence or absence of a suitable audience. We investigated effects on alarm calling by presenting overhead predator models to cockerels in the presence of a variety of social companions. The production of aerial predator calls in response to hawk silhouettes varied with the presence or absence of a member of the same species. The kinds of audience investigated included the mate, unfamiliar females, other females and males with which subjects had had prior visual and auditory contact, and broody hens with and without young. Domestic chicks, unrelated to the subjects, were almost as effective an audience as conspecific adults. A member of another species, however, failed to potentiate alarm-call production. The subjects gave more alarm calls when they were in the presence of either a male or a female audience than when they were alone. By manipulating the visibility of overhead predator models to the subjects and to the audience, we showed that the subjects were not cued by alarm and escape behaviors of the audience. Comparisons with food calling indicate that, in deciding whether to emit a signal in response to the appropriate referent (e.g., food or predators), chickens respond to subtle differences in the nature of the audience with behaviors that vary from one communicative context to another.